Our previous studies on the mechanism of transformation by retroviral oncogenes have indicated that there are fundamental differences in monovalent cation transport in NIH/3T3cells and other cell lines following transformation by the oncogene ras. These differences are "corrected" in flat revertant cells that resist transformation by ras and certain other oncogenes. Using sodium uptake and pH measurements, we have now shown that ras- transformed cells do indeed show altered sodium/hydrogen antiport activity when compared to controls. The mechanisms by which revertant cells require resistance to ouabain is under investigation. The sodium/hydrogen antiporter is known to be stimulated by transforming growth factor alpha and may play a critical role in the control of cell division. We are continuing to isolate new cellular mutants that are resistant to transformation by retroviral oncogenes. The proline analog 4-cis-hydroxyproline is less toxic for normal cells than for transformed cells. We have demonstrated that cis-hydroxyproline can be used to isolate nontransformed (revertant) cells from populations of both ras and raf transformed cells. The properties of these revertants are now being compared with those of the initial ras revertants isolated following treatment with ouahain. Retroviral vectors have many advantages over other DNA transfer techniques, including a stahle integration of single copies of the gene of interest and introduction into a wide variety of cells with a high transfer frequency. We are now in the process of constructing new retroviral vectors containing oncogenes so that we can engineer cells useful for the isolation of a second generation of transformation resistant cells.